Abstract
Real-time polymerase chain reaction (PCR) is the standard for nucleic acid detection and plays an important role in many fields. A new chip design is proposed in this study to avoid the use of expensive instruments for hydrophobic treatment of the surface, and a new injection method solves the issue of bubbles formed during the temperature cycle. We built a battery-powered real-time PCR device to follow polymerase chain reaction using fluorescence detection and developed an independently designed electromechanical control system and a fluorescence analysis software to control the temperature cycle, the photoelectric detection coupling, and the automatic analysis of the experimental data. The microchips and the temperature cycling system cost USD 100. All the elements of the device are available through open access, and there are no technical barriers. The simple structure and manipulation allows beginners to build instruments and perform PCR tests after only a short tutorial. The device is used for analysis of the amplification curve and the melting curve of multiple target genes to demonstrate that our instrument has the same accuracy and stability as a commercial instrument.
Highlights
Polymerase chain reaction (PCR) is a molecular reaction used to amplify a specific segment of DNA [1,2,3]
PCR plays an important role in many fields, including pathogen diagnosis, bioprospecting, and environmental protection [4,5,6,7,8,9,10,11,12]
Many commercial PCR instruments, including the Applied Biosystems (ABI) PRISM® 7900HT and Bio-Rad iQ5 systems, have been successfully brought to the market. These instruments have a good detection performance, but real-time PCR is almost 10 times more expensive than ordinary PCR platforms due to its high sensitivity and signal reporting method [19,20,21,22,23]. The cost of these commercial quantitative PCR devices is relatively high, which is an issue for resource-limited areas
Summary
Polymerase chain reaction (PCR) is a molecular reaction used to amplify a specific segment of DNA [1,2,3]. Most devices need an external power supply which greatly limits their use For these reasons, many researchers are developing low-cost real-time fluorescent quantitative PCR systems [23,24]. Polymerase chain reaction requires a denaturation temperature of 95 ◦C to uncoil the double-stranded DNA, which is very close to the boiling point of water This causes bubbles to form in the microfluidic chips and the reaction to fail [25,26]. These chips need to be used as soon as possible after processing, which limits their application [23,29,30] Another challenge is to develop a control system with no technical barriers and as little hardware as possible to achieve the coupling between the temperature cycle, the optical imaging, and the automatic analysis of the corresponding data.
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